1. Field of the Invention
The present invention relates to uses of immunological reagents specific for a human transmembrane efflux pump protein (P-glycoprotein). Specifically, the invention relates to uses of such immunological reagents that specifically recognize P-glycoprotein that is in a biochemical conformation adopted in the presence of certain cytotoxic, lipophilic drugs that are substrates for P-glycoprotein, in the presence of cellular ATP depleting agents, and by certain mutant embodiments of Pgp. In particular, the invention provides methods of using such immunological reagents for anticancer drug screening and development.
2. Background of the Invention
Many human cancers express intrinsically or develop spontaneously resistance to several classes of anticancer drugs, each with a different structure and different mechanism of action. This phenomenon, which can be mimicked in cultured mammalian cells selected for resistance to certain plant alkaloids or antitumor antibiotics such as colchicine, vinblastine and doxorubicin (formerly known as Adriamycin), is generally referred to as multidrug resistance (xe2x80x9cMDRxe2x80x9d; see Roninson (ed)., 1991, Molecular and Cellular Biology of Multidrug Resistance in Tumor Cells, Plenum Press, N.Y., 1991; Gottesman et al., 1991, in Biochemical Bases for Multidrug Resistance in Cancer, Academic Press, N.Y., Chapter 11 for reviews). The MDR phenotype presents a major obstacle to successful cancer chemotherapy in human patients.
MDR frequently appears to result from decreased intracellular accumulation of anticancer drugs as a consequence of increased drug efflux related to alterations at the cellular plasma membrane. When mutant cell lines having the MDR phenotype are isolated, they are found to express an ATP-dependent non-specific molecular xe2x80x9cpumpxe2x80x9d protein (generally known as P-glycoprotein) that is located in the plasma membrane and keeps the intracellular accumulation of an anti-cancer drug low enough to evoke the drug-resistance phenotype. This protein (which has been determined to be the gene product of the MDR1 gene in humans) facilitates active (i.e., energy-dependent) drug efflux from the cell, against a concentration gradient of (generally) lipophilic compounds, including many cytotoxic drugs.
The gene encoding P-glycoprotein (which is also known as gp170-180 and the multidrug transporter) has been cloned from cultured human cells by Roninson et al. (see U.S. Pat. No. 5,206,352, issued Apr. 27, 1993), and is generally referred to as MDR1. The proteinproduct of the MDR1 gene, most generally known as P-glycoprotein (xe2x80x9cPgpxe2x80x9d), is a 170-180 kilodalton (kDa) transmembrane protein having the aforementioned energy-dependent efflux pump activity.
Molecular analysis of the MDR1 gene indicates that Pgp consists of 1280 amino acids distributed between two homologous halves (having 43% sequence identity of amino acid residues), each half of the molecule comprising six hydrophobic transmembrane domains and an ATP binding site within a cytoplasmic loop. Only about 8% of the molecule is extracellular, and carbohydrate moieties (approximately 30 kDa) are bound to sites in this region (Chen et al., 1986, Cell 47: 381-387).
Expression of Pgp on the cell surface is sufficient to render cells resistant to many (but not all) cytotoxic drugs, including many anti-cancer agents. Pgp-mediated MDR appears to be an important clinical component of drug resistance in tumors of different types, and MDR 1 gene expression correlates with resistance to chemotherapy in different types of cancer.
Pgp is also constitutively expressed in many normal cells and tissues (see Cordon-Cardo et al., 1990, J. Histochem. Cytochem. 38: 1277; and Thiebaut et al., 1987, Proc. Natl. Acad. Sci. USA 84: 7735 for reviews). In hematopoietic cells, Neyfakh et al. (1989, Exp. Cancer Res. 185: 496) have shown that certain subsets of human and murine lymphocytes efflux Rh123, a fluorescent dye that is a Pgp substrate, and this process can be blocked by small molecule inhibitors of Pgp. It has been demonstrated more recently that Pgp is expressed on the cell-surface membranes of pluripotent stem cells, NK cells, CD4- and CD8-positive T lymphocytes, and B lymphocytes (Chaudhary et al., 1992, Blood 80: 2735; Drach et al., 1992, Blood 80: 2729; Kimecki et al., 1994, Blood 83: 2451; Chaudhary et al., 1991, Cell 66: 85). Pgp expression on the cell surface membranes of different subsets of human lymphocytes has been extensively documented (Coon et al., 1991, Human Immunol. 32: 134; Tiirikainen et al., 1992, Ann. Hematol. 65: 124; Schluesener et al., 1992, Immunopharmacology 23: 37; Gupta et al., 1993, J. Clin. Immunol. 13: 289). Although recent studies suggest that Pgp plays a role in normal physiological functions of immune cells (Witkowski et al., 1994, J. Immunol. 153: 658; Kobayashi et al., 1994, Biochem. Pharmacol. 48: 1641; Raghu et al., 1996, Exp. Hematol. 24: 1030-1036), the physiological role of Pgp in normal immune cells has remained unclear to date.
Once the central role in MDR played by Pgp was uncovered, agents with a potential for reversing MDR phenotypes were developed that target Pgp. Several classes of drugs, including calcium channel blockers (e.g., verapamil), immunosuppresants (such as cyclosporines and steroid hormones), calmodulin inhibitors, and other compounds, were found (often fortuitously) to enhance the intracellular accumulation and cytotoxic action of Pgp-transported drugs (Ford et al., 1990, Pharm. Rev. 42: 155). Many of these agents were found to inhibit either drug binding or drug transport by Pgp (Akiyamaetal., 1988, Molec. Pharm. 33: 144; Horio et al., 1988, Proc. Natl. Acad. Sci. USA 84: 3580). Some of these agents themselves were found to bind to and be effluxed by Pgp, suggesting that their enhancing effects on the cytotoxicity of Pgp substrates are due, at least in part, to competition for drug binding sites on this protein (Cornwell et al., 1986, J. Bio. Chem. 261: 7921; Tamai, 1990, J. Biochem. Molec. Biol. 265: 16509).
Many of these agents, however, also have strong, deleterious side effects at physiologically-achievable concentrations. These systemic side effects severely limit the clinical use of these agents as specific inhibitors of Pgp or for negative selection against Pgp-expressing tumor cells. Most of the known MDR-reversing drugs used in clinical trials have major side effects unrelated to inhibition of Pgp, such as calcium channel blockage (verapamil) or immunosuppression (cyclosporines and steroids). Similarly, targeting of cytotoxic drugs to Pgp-expressing cells is capable of compromising normal tissue function in normal cells (such as kidney, liver, colonic epithelium, etc.) that normally express Pgp. These drawbacks restrict the clinically-achievable dose of such agents and ultimately, their usefulness.
Immunological reagents also provide a means for specifically inhibiting drug efflux mediated by Pgp. Monoclonal antibodies specific for Pgp are known in the art.
Hamada et al., 1986, Proc. Natl. Acad. Sci. USA 83: 7785 disclose the mAbs MRK-16 and MRK-17, produced by immunizing mice with doxorubicin-resistant K-562 human leukemia cells. MRK-16 mAb was also reported to modulate vincristine and actinomycin D transport in resistant cells, and MRK-17 was shown to specifically inhibit growth of resistant cells with these drugs.
Meyers et al., 1987, Cancer Res. 49: 3209 disclose mAbs HYB-241 and HYB-612, which recognize an external epitope of Pgp.
O""Brien et al., 1989, Proc. Amer. Assoc. Cancer Res. 30: Abs 2114 disclose that mAbs HYB-241 and HYB-612 increased the accumulation of vincristine and actinomycin D in tumor cells and increased the cytotoxicity of combinations of these drugs with verapamil.
Tsuruo et al., 1989, Jpn. J. Cancer Res. 80: 627 reported that treatment of athymic mice that had been previously inoculated with drug resistant human ovarian cancer cells with the mAb MRK16 caused regression of established subcutaneous tumors.
Hamada et al., 1990, Cancer Res. 50: 3167 disclosed a recombinant chimeric antibody that combines the variable region of MRK-16 with the Fc portion of a human antibody, and showed this chimeric antibody to be more effective than MRK-16 mAb in increasing cytotoxicity in vitro.
Pearson et al., 1991, J. Natl. Cancer Inst. 88: 1386 disclosed that MRK-16 mAb increased the in vivo toxicity of vincristine to a human MDR colon cancer cell line grown as a xenograft in nude mice. The in vitro potentiation of drug cytotoxicity by MRK-16 mAb was, however, weak relative to known chemical inhibitors of Pgp action, and was apparently limited to only two Pgp substrates (vincristine and actinomycin D), having no effect on cytotoxicity by doxorubicin.
Cinciarelli et al., 1991, Int. J. Cancer 47: 533 disclosed a mouse IgG2a mAb, termed MAb657, having cross reactivity to Pgp-expressing human MDR cells. This mAb was shown to increase the susceptibility of MDR cells to human peripheral blood lymphocyte-mediated cytotoxicity, but was not shown to have an inhibitory effect on the drug efflux activity of Pgp.
Arcesi et al., 1993, Cancer Res. 53: 310-317 disclosed mAb 4E3 that binds to extracellular epitopes of Pgp but does not disrupt drug efflux or potentiate MDR drug-induced cytotoxicity.
Mechetner and Roninson, U.S. Pat. No. 5,434,075, issued Jul. 18, 1995, disclose mAb UIC2, having specificity for extracellular Pgp epitopes. This antibody was also shown to effectively inhibit Pgp-mediated drug efflux in MDR cells, and to reverse the MDR phenotype in vitro thereby, for a number of structurally and functional different cytotoxic compounds and all tested chemotherapeutic drugs known to be substrates for Pgp-mediated drug efflux.
There is a need in the art to develop new Pgp inhibitors for preventing or overcoming multidrug resistance in human cancer. In developing new pharmaceuticals, it is essential to determine whether a drug candidate is a Pgp substrate and is effluxed by Pgp expressed in normal or tumor cells. This is important because, on the one hand, such drugs are expected to inhibit Pgp expression in normal cells (in the gastrointestinal tract, excretory organs like kidney, certain hematopoietic cells and the blood-brain and testicular barriers), as well as tumor cells, and to compromise normal function in such organs thereby. On the other hand, tumors derived from such Pgp-expressing tissues are frequently intrinsically multidrug resistant and therefore unaffected by chemotherapeutic intervention. Finally, in all multidrug resistant tumor cells, anti-cancer drugs transported by Pgp decrease intracellular drug concentration, reduce the drug""s xe2x80x9ctherapeutic windowxe2x80x9d and ultimately reduce the effectiveness of chemotherapeutic treatment. Thus, there is a great need in the art for reagents and assays that permit the rapid, efficient and economical screening and development of effective Pgp inhibitors.
It has been shown that small molecules that are transported by Pgp can be used to competitively inhibit Pgp-mediated efflux of chemotherapeutic drugs that are Pgp substrates. Inhibition of cytotoxic drug efflux from tumor cells in the presence of small molecule Pgp inhibitors has been shown to increase intracellular concentrations of drug and thereby increase its cytotoxic effectiveness. Such small molecules are considered promising drug candidates for selective potentiation of the antitumor effects of several anticancer drugs, including doxorubicin, taxol, vinblastine and VP-16. For example, recent clinical trials of a (relatively) non-toxic cyclosporin analog (PSC833, Novartis Corp.) demonstrated the feasibility of using small molecule Pgp inhibitors for reversing multidrug resistance in patients with hematological malignancies. These results are being actively pursued by a variety of pharmaceutical and biotechnology companies and academic researchers. Thus, development of inexpensive and reliable tests for high throughput screening and identification of new Pgp substrates is important for the development of potent Pgp reversing agents.
At present there are two techniques available for identifying Pgp transport substrates. The first is a dye-efflux assay performed using flow cytometry and is based on competitive inhibition of Pgp-mediated efflux of fluorescent dyes such as rhodamine 123. The second is an in vitro cytotoxicity assay that uses the ability of Pgp substrates to competitively inhibit Pgp-mediated efflux of cytotoxic drugs in Pgp-expressing multidrug resistant cells. In this assay, competitive inhibition of Pgp in cells cultured in the presence of cytotoxic concentrations of Pgp-effluxed cytotoxic drugs results in increased intracellular concentration of such drugs and decreased cell growth. Both assays suffer from the disadvantage that they are laborious and time-consuming and are not suitable for high throughput screening or clinical laboratory testing. In addition, these assays are not specific for Pgp because fluorescent dyes and cytotoxic drugs are also transport substrates for related multidrug resistance transporters (such as MRP; Grant et al., 1994, Cancer Res. 54: 357-361)
There remains a need in the art for a rapid, reliable, efficient and inexpensive method for high throughput screening of compounds for Pgp inhibiting activity in order to develop more effective chemotherapeutic treatment of human cancer patients.
The invention also provides methods for evaluating novel cytotoxic, chemotherapeutic drugs and Pgp inhibitors. The methods of the invention are based on the development of novel immunological reagents specific for Pgp in a biochemical conformation adopted in the presence of Pgp-mediated transport substrates or ATP depleting agents. The capacity to discriminate between compounds that induce this conformation in Pgp and those that do not provides a way to identify Pgp inhibitors that can be used in high throughput screening assays. These methods can be used as screening assays based on enhanced binding of certain immunological reagents such as UIC2 mAb (A.T.C.C. Accession No. HB 11027) or its derivatives in the presence of Pgp substrates and enable rapid, reliable and cost-effective characterization of potential new Pgp-targeted drugs.
The methods of the invention comprise the steps of contacting a mammalian cell expressing Pgp with an immunological reagent such as UIC2 mAb in the presence and absence of a putative Pgp binding substrate and comparing binding of the immunological reagent in the presence of the test compound with immunological reagent binding in the absence of the test compound. In preferred embodiments, the immunological binding agent is detectably labeled. More preferably, the immunological reagent is detectably labeled with a fluorescent label, and binding affinity is detected by fluorescence activated cell sorting (FACS), immunohistochemistry and similar staining methods. In one aspect of the methods of the invention, Pgp expression levels are determined, providing the capacity to quantitatively compare results between assays. In a second aspect, enhanced binding activity of the immunological reagents provide a way of determining Pgp binding capacity of the test compound. The assays of the invention thus advantageously provide information on both Pgp expression and function simultaneously.
An additional advantage of the methods of the invention is that the use of immunological reagents specific for Pgp reduces the possibility that the assay results contain contributions from related species involved in multidrug resistance, such as MRP.
Specific preferred embodiments of the present invention will become evident from the following more detailed description of certain preferred embodiments and the claims.